1. Field of the Invention
The present invention relates to the production of L-threonine involving microorganisms. More particularly, the present invention relates to a process for producing L-threonine with a high yield, in which additional one or more copies of the phosphoenolpyruvate carboxylase (ppc) gene and the threonine operon are inserted into a particular site of the chromosomal DNA of a microorganism, while its inherent ppc gene and threonine operon remain, to increase the expression of the ppc gene encoding an enzyme to convert phosphoenolpyruvate to oxaloacetate, which is a threonine biosynthetic precursor, and the expression of genes encoding enzymes engaged in the synthetic pathway of threonine from oxaloacetate, such as aspartokinasel-homoserine dehydrogenase (thrA), homoserine kinase (thrB), and threonine synthase (thrC).
2. Description of the Related Art
L-threonine, a kind of essential amino acid, is widely used as an additive to animal fodder and food, and as fluids and synthetic materials for medical and pharmaceutical use. L-threonine is produced by fermentation using synthetic mutants derived from wild types of Escherichia coli, Corynebacterium, Serratia, and Providencia. These variant strains are known to include amino acid analogs, pharmaceutical-resistant mutants, and synthetic pharmaceutical-resistant mutants rendered auxotrophic for diaminopimelic acid, methionine, lysine, or isoleucine (Japanese Laid-open Patent Application No. hei 2-219582, Appl., Microbiolo. Biotechnol., 29, 550-553 (1988), and Korean Patent Publication No. 92-8365).
A common approach to increase the level of expression of a particular gene uses a plasmid that gives a greater copy number to a microorganism in order to increase the number of genes in the microorganism (Sambrook et al., Molecular cloning, Second Edition, 1989, 1.3-1.5). A target gene is integrated into a plasmid, and the host microorganism is transformed with the recombinant plasmid to cause an increase in the number of genes in the host microorganism according to the copy number in the plasmid. A partial success in this type of approach to improve threonine productivity is reported in U.S. Pat. No. 5, 538,873. However, most technologies using such recombinant plasmids overexpress a particular gene, which is undesirable for the host microorganism, and causes a problem of plasmid instability so that the plasmid is lost during cultivation of the recombinant strain.
To address this problem, approaches to add antibiotics to culture media or to use an expression regulatory plasmid were suggested (Sambrook et al. Molecular cloning, Second Edition, 1989, 1.5-1.6 & 1.9-1.11). In using the expression regulatory plasmid to yield a particular product, cell cultivation is performed under non-expression conditions in the growth stage to reduce a load to the host microorganism and temporary expression is induced after full growth of the microorganism. However, most expression regulatory plasmids target protein synthesis. Producing primary metabolites is closely associated with the growth of microorganisms, so it is difficult to increase the yield of the primary metabolites unless target genes are expressed in the growth stage. The production of threonine, a primary metabolite, is such a case.
As an effort to compensate for this drawback, a particular threonine biosynthetic gene was incorporated into a chromosomal DNA to produce threonine (U.S. Pat. No. 5,939,307). However, this approach replaces a chromosomal gene by an inducible promoter-substituted gene, which is hardly expected to markedly increase the expression of the threonine operon gene.
Therefore, unlike the conventional substitution method, the present inventors have inserted an additional ppc gene and threonine operon into a particular site (lacZ gene) of the chromosomal DNA while the original chromosomal gene of a host microorganism remains, and found that it provides dual effects as a result of the original chromosomal gene and the inserted ppc gene and threonine operone. Most current genetic engineering techniques applied to increase the yield of threonine are focused on the biosynthetic pathway, starting with oxaloacetate. However, the present invention involves also ppc, which is an oxaloacetate inducer enzyme acting in the preceding step, as well as the threonine biosynthetic enzymes to purposely guide the flow of carbons from phosphoenolpyruvate into the oxaloacetate synthetic pathway. The present invention also allows insertion of two or more copies of gene if necessary.